This is an amended application to study a novel function for the inducible enzyme that catalyzes heme degradation, heme oxygenase-1 (HO-1), in the regulation of mitochondrial biogenesis. The proposed mechanism is based on the endogenous production of CO by HO (HO/CO). CO, like nitric oxide (NO), is increasingly recognized as a gaseous signaling molecule serving regulatory roles in health and disease. We have discovered that CO at physiological concentrations up-regulates the nuclear transcription factors, nuclear respiratory factors (NRF) -1 and -2, and the central co-activator, PGC-11, which regulate , mitochondrial biogenesis. Our preliminary data show that CO promotes mtDNA replication and increases mtDNA copy number in the mouse heart during mitochondrial biogenesis. The latter is an essential process under nuclear control that requires mitochondrial fusion, fission, and respiratory protein synthesis in order to meet the organ's continuous demand for aerobic ATP synthesis for contractile function. The pathways by which HO/CO promotes biogenesis are not yet well defined but we have new preliminary data implicating CO-cytochrome c oxidase a3-heme binding in mitochondria in the mechanism, leading to H2O2-mediated activation of the pro-survival kinase, Akt/PKB and nuclear translocation of the redox-sensitive Nrf2 transcription factor. Our hypothesis is that physiological (endogenous) CO produced by HO serves a cell survival function by redox activation of mitochondrial biogenesis to produce an anti-oxidant and anti- apoptotic mitochondrial phenotype. We propose three Specific Aims: Aim 1: Test the hypothesis that exogenous and endogenous CO activates cardiac mitochondrial biogenesis through Akt-dependent phosphorylation of PGC-11. Aim 2: Test the hypothesis that mitochondrial H2O2 signaling by HO/CO and its interplay through the Nrf2 transcription factor regulate HO-1 and NRF-1 gene expression for the transcriptional regulation of mitochondrial biogenesis. Aim 3: Test the hypothesis that the myocardial protective effect of CO depends on endogenous HO-1 activity and the generation of an apoptosis-resistent mitochondrial phenotype in doxorubicin cardiomyopathy. The completion of these Aims will expand and develop our understanding of the role of CO as a cell-signaling molecule in mitochondrial health and disease. The implication is that HO/CO-regulated mitochondrial biogenesis is fundamental to the maintenance of normal cardiovascular function as well as to adaptation to oxidative stress and pathogenic inflammation. This would provide a unifying mechanism for the protective role of HO- 1 that may be amenable to therapeutic intervention by a range of unique and novel strategies. PUBLIC HEALTH RELEVANCE: This is new proposal to study a novel function for heme oxygenase-1 (HO-1), one of two main isoforms of the enzyme that converts heme into biliverdin, Fe, and carbon monoxide (CO). Our preliminary data implicate HO-1, through the production of CO, as a regulator of mitochondrial biogenesis. Our work in the mouse heart and in cardiomyocytes suggests the hypothesis that HO/CO-regulated mitochondrial biogenesis is fundamental for adaptation to oxidative and inflammatory stress. A successful test of our hypothesis would establish a unifying mechanism for the diverse protective roles of HO-1 in health and disease. [unreadable] [unreadable] [unreadable]